[Histonet] Giemsa staining on Bone Marrow smears

[McLaughlin, Sharon R]

Hi,

 

This is in response to a question about the Giemsa staining on bone
marrow smears.  First of all, the procedure for making the smears has to
be ideal or it will not be the best staining.  Make sure whomever is
doing the smears is using the right technique.  That can make all the
difference in the world.

 

I use the May-Gruwald Giemsa Stain from R.D. Lillie.

 

Reagents:  

 

Stock Giemsa Solution:

Giemsa powder .............................................1.0 gm  

Glycerin......................................................66.0 ml

Absolute Methanol....................................... 66.0 ml

*********Mix the giemsa powder and the glycerin and place in a 60 degree
oven for 2 hours.  After the incubation, add the 66 ml of absolute
methanol.  Mix well.

 

Working Giemsa Solution:

Stock Giemsa Solution.................................40 drops

Distilled water.............................................40 ml

*********Make fresh, do not reuse

 

1% Glacial Acetic Acid:

Concentrated Glacial Acetic Acid..................1.0 ml

Distilled water.......................................   99.0 ml

 

Stock Jenner:

Jenner stain, dry powder.............................1.0 gm

Absolute methanol.................................400.0 ml

 

Working Jenner:

Stock Jenner solution...............................25.0 ml

Distilled water.........................................25.0 ml

 

Procedure:

 

1.	Deparaffinize and hydrate to tap water.
2.	Wash in running water for 10 minutes.
3.	Rinse in distilled water, 2 changes.
4.	Place in absolute methyl alcohol, 2 changes, 3 minutes each.
5.	Place in working Jenner solution for 6 minutes.
6.	Place in working Giemsa solution for 45 minutes.
7.	IMPORTANT STEP:

Handle each slide individually in this and subsequent steps.  Rinse
quickly in distilled water - 1 dip.

8.	Differentiate in 1% glacial acetic acid just until the sections
becomes pink!!!!!!!.  Check microscopically for well differentiated
nuclei.  Red cells will become reddish pink.
9.	Rinse well in several changes in distilled water.
10.	Dehydrate quickly, clear and mount.

 

 

Results:

 

Mast cell granules.....................................Rust red

Nuclei.................................................... Blue

Cytoplasm...............................................Pink to rose


Bacteria and spirochetes............................Rose to purple

Rickettsiae............................................. Violet

Chromatin of parasites.............................. Red

RBC's................................................... Salmon red